The mitochondria of cardiac muscle use 95-98% of the cellular oxygen consumed and synthesize the principal source of energy for heart cell function, ATP via oxidative phosphorylation. This occurs in the mitochondrial inner membrane and is mediated by a series of electron transfer proteins. Cytochrome c oxidase is the terminal electron transfer enzyme in mitochondria that reduces molecular oxygen and conserves the energy of its electron transport by the translocation of a proton across the inner membrane. The subunit structure of the enzyme is well characterized, however, the subunit that is responsible for proton translocation across inner membrane is unknown. The goal of this project is to delineate which of the subunits of cytochrome c oxidase is responsible for this proton translocation. Experiments are proposed that will remove one subunit (III) from the enzyme, test for proton translocation activity, and attempt to restore that activity by adding back the purified subunit (III) in a reconstituted system. The surface topography of the enzyme deficient in subunit III will be mapped by the use of water soluble radioactive chemical modification reagents. Intersubunit interactions in the subunit III deficient enzyme will be examined by the use of cleavable, bifunctional chemical cross linking reagents. The proton translocating properties of subunit III of cytochrome c oxidase will be studied and directly compared to the proton translocating subunit of the ATP synthetase. The amino acid functional groups responsible for proton translocation and the mechanism of proton translocation will be investigated in both subunits. The structural domain responsible for proton translocation in each subunit will be isolated and reconstituted in order to investigate similarities in the mechanism of proton translocation in each subunit. The dependence of the conformation of cytochrome c oxidase on its oxidation-reduction state will be explored to determine if changes in protein structure are important in the energy conservation mechanism.